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A 45-year-old woman was admitted because of hypertension and hypokalemia. Primary amenorrhea from birth was noted. Plasma renin activity (PRA), 17alpha-hydroxyprogesterone and androgen levels were low, but progesterone, 11-deoxycorticosterone, corticosterone and adrenocorticotropic hormone (ACTH) were elevated, resulting in a diagnosis of 17alpha-hydroxylase deficiency. Abdominal magnetic resonance imaging revealed a round mass in the left adrenal region, the specimen of which was diagnosed as myelolipoma. After removal of the tumor, the blood pressure, serum potassium and hormone levels were unchanged, indicating an adrenal non-functioning tumor. Excessive ACTH secretion over a long period may stimulate the development of adrenal myelolipoma.

Telomerase, the ribonucleoprotein enzyme that elongates chromosomal ends, or telomeres, is repressed in most normal somatic cells but reactivated in transformed cells to compensate for the progressive erosion of the telomeres during cell divisions. In accordance with this hypothesis, the presence of telomerase activity has been reported in more than 90% of human cancers, whereas most normal tissues or benign tumors contain low or undetectable telomerase activity. Reactivation of telomerase has also been widely reported in endocrine neoplasms and in hormone-related cancers. In the present study, we review the most recent publications on telomerase in these types of tumors. The hormonal regulation of telomerase activity and the possible strategies for cancer therapy based on the inhibition of telomerase has also been discussed.

Background: 11beta-hydroxysteroid dehydrogenase Type-2 (11beta-HSD2) is an unidirectional enzyme that catalyzes the conversion of glucocorticoid hormones cortisol and corticosterone (B) into their corresponding inactive forms, cortisone, and 11-dehydrocorticosterone (DH-B). We have provided evidence that 11beta-HSD2 is expressed as messenger RNA (mRNA) and protein in human adrenocortical cells, where its activity is inhibited in vitro by the main glucocorticoid agonists, adrenocorticotropic hormone (ACTH) and angiotensin-II. It seemed worthwhile, therefore, to study the gene expression and activity of 11beta-HSD2 in cortisol-secreting adrenocortical adenomas., Methods: Three adrenal adenomas that produced Cushing syndrome were recruited. Three normal adrenal glands were obtained from patients who underwent unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. 11beta-HSD2 gene expression was studied by reverse transcriptionpolymerase chain reaction (RT-PCR) in adenoma and normal adrenocortical tissue. Cortisol, B, cortisone, and DH-B production by adenoma and adrenal slices in vitro was assayed by quantitative high-performance liquid chromatography (HPLC), and the activity of 11beta-HSD2 was evaluated by measuring the conversion of [3H]-cortisol to [3H]-cortisone., Results: RT-PCR allowed the detection of the 11beta-HSD2 mRNA in the three adrenal adenomas and normal adrenal cortices examined. Under basal conditions, adenoma slices secreted higher amounts of cortisol and B, but markedly lower amounts of cortisone and DH-B than adrenal slices. ACTH raised cortisol and B production from both specimens, and it lowered cortisone and DH-B yield. The level basal conversion of [3H]-cortisol to [3H]-cortisone was notably less in adenomas than in adrenals, and ACTH decreased it in both tissues., Conclusions: Collectively, our findings indicate that cortisol-secreting adrenal adenomas express the 11beta-HSD2 gene, but the activity of the enzyme is suppressed in adenomas when compared with the normal adrenal cortex. We advance the hypothesis that the elevated local concentration of steroid hormones that occur in adenomas down-regulates 11beta-HSD2 activity, thereby contributing to their abnormal steroidogenic function.

We tested the effects of 17beta-estradiol as well as its catechol- and methoxy-derivatives, two androgens (DHEA and testosterone), a glucocorticoid (cortisol), a mineralocorticoid (aldosterone), and progesterone on the activity of ERK(1,2), a key component of the ERK/MAPK enzyme phosphorylation cascade, in SW-13 human adrenal carcinoma cells. After a 24-hour exposure SW-13 cells incubated with 10(-5) M concentrations of 17beta-estradiol, its 2-hydroxy or its 2-methoxy derivative, all had elevated ERK activities (196%, 159%, and 275%, respectively) relative to control cells (p < 0.01). Incubation with testosterone resulted in 162% of control ERK activity (p < 0.01), whereas incubation with the far weaker androgen DHEA or with cortisol, aldosterone, or progesterone had no significant effects. These findings suggest sex steroid-specific influences in the induction or activation of signal transduction pathways known to play a crucial role in cellular proliferation and differentiation.

A 7-yr-old girl presented with isosexual precocious puberty secondary to a feminizing adrenal adenoma. The adrenal tumor was found to express aromatase messenger ribonucleic acid. Enzyme kinetic studies revealed a high level of aromatase activity in the adrenal tumor, with a K(m) of 45 nmol/L and a maximum velocity of 25.6 pmol/mg.h. Aromatase activity was approximately 500-fold higher in the tumor than in adjacent normal adrenal tissue. Although histopathological examination of the tumor was most consistent with a benign adenoma, the aromatase transcripts present in the tumor corresponded to those previously associated with malignant as well as benign tumors. We consider the pattern of aromatase expression sufficient to warrant continued follow-up for tumor recurrence. Our case demonstrates that isosexual precocious puberty secondary to a feminizing adrenal tumor can be due to estrogen synthesis from the tumor itself rather than peripheral aromatization as had been previously theorized.

The aim of the study is to evaluate MMP-1, MMP-8 and MMP-9 serum levels in patients with adrenal tumors prior to and after surgery. Metalloproteinase-1 (MMP-1), MMP-8 and MMP-9 serum levels were evaluated in 43 patients operated on at our clinic between 1997-1999. Forty-one (95.3%) patients underwent adrenalectomy. Two (4.7%) patients were disqualified from surgery due to infiltration of adjacent tissues. MMP-1, MMP-8 and MMP-9 serum levels were determined at the admission and in case of surgery again one month after the operation. ELISA assay (K&D) was applied. Tumor type was determined on the basis of clinical, hormonal and histopathological examination. The correlation between MMP levels and tumor sizes was also evaluated. Patients were divided into 6 groups. Group I included 11 patients with adrenocortical carcinoma (4 with Cushing's syndrome and 7 with incidentalomas); group II--6 patients with benign hormonally active adrenocortical adenoma (4 with Cushing's syndrome and 2 with Conn's syndrome); group III--patients with benign, hormonally inactive adenocortical adenoma; group IV--6 patients with benign, hormonally active phaeochromocytoma; group V--4 patients with hormonally inactive phaeochromocytoma; group VI--5 patients with hormonally inactive adrenal tumors of extraglandular origin (2 myolipomas, 2 fibrolipomas, 1 hammartoma). The control group comprised 10 healthy individuals. Increased MMP-8 and MMP-9 levels were noted in patients with benign and malignant adrenal tumors. No increase of MMP levels was found in patients with tumors of extraglandular origin. The increased MMP-8 and MMP-9 levels occurred most frequently in patients with adrenocortical and hormonally active adrenomedullar cancer, and most rarely in patients with hormonally active adrenocortical tumors. MMP-8 and MMP-9 serum levels did not significantly differ between patients with adrenocortical incidentaloma cancers and in patients with benign incidentalomas. MMP-8 and MMP-9 levels were not increased in patients with inoperable adrenocortical cancers. Serum MMP-1 levels were not increased in patients with benign and malignant adrenal tumors. After surgery, MMP-8 and MMP-9 levels decreased significantly in patients with adrenocortical cancers, whereas the decrease of these MMPs in patients with benign tumors, although noticeable, was not statistically significant. MMP-8 and MMP-9 levels decreased significantly in all patients with increased preoperative levels, although they remained higher than the maximum normal values only in few patients (in 7 and 2 patients, respectively). No correlation between the levels of evaluated MMPs and tumor sizes were found.

We examined clinical, endocrinological and molecular biological aspects of an estrogen-secreting adrenal carcinoma in an 18-month-old male to clarify the pathogenesis of this condition. An 18-month-old boy was referred for evaluation of progressive bilateral gynecomastia and appearance of pubic hair. The patient had elevated plasma estradiol (349 pg/ml) and testosterone (260 ng/dl) levels that completely suppressed FSH and LH levels, and was subsequently diagnosed with an adrenal tumor on the right side. After removal of a 300-g adenocarcinoma, gynecomastia regressed and essentially normal hormone levels were restored. Aromatase activity in the tumor tissue determined by the 3H-water method was 71.0-104.4 pmol/min/mg protein. High levels of aromatase protein and mRNA in the tumor tissue were also demonstrated, while neither aromatase activity nor protein was detected in normal adrenal glands. To investigate the regulation of aromatase expression in the adrenal carcinoma, we examined the usage of alternate promoters responsible for aromatase gene transcription. In the present case, the amounts of aromatase mRNA utilizing gonadal types of exon 1c (1.3) and 1d (II) were significantly higher than those that using other exon 1s. This result suggested that the utilization of a gonadal-type exon 1 might be involved in the over-production of aromatase in estrogen-secreting adrenal carcinoma.

Inducible nitric oxide synthase (iNOS) has been reported in tangle-bearing neurons of patients with Alzheimer's disease (AD), and can be induced by tumor necrosis factor-alpha (TNFalpha). High CNS levels of TNFalpha are associated with neurodegenerative diseases such as AD, where neurons dependent on neurotrophins such as nerve growth factor (NGF) are particularly affected. In this study we determined the effect of TNFalpha on iNOS in NGF-responsive pheochromocytoma (PC12) cells. We found that while TNFalpha and NGF alone were unable to induce iNOS, their simultaneous addition resulted in iNOS induction and the release of nitric oxide. Our results suggest that synergistic iNOS induction by TNFalpha and NGF may occur in selective population of NGF-responsive neurons in the presence of elevated CNS levels of TNFalpha.

Objective: To report the occurrence of a giant left adrenal tumor and bilateral testicular masses (adenomatous hyperplasia of Leydig cells) in a young man with congenital adrenal hyperplasia due to 21-hydroxylase deficiency., Methods: The clinical, radiologic, endocrinologic, and pathologic features of this case are correlated with the findings in the literature., Results: The interesting elements in this case are the rare pathologic features of the left adrenal lesion (pigmented adrenal hyperplasia with myelolipomatous changes) and the association with the infrequent testicular adrenal rest tumors. The absence of enlargement of the right adrenal gland was unexplained., Conclusion: The presence of these two rare complications seemed to be associated with poor adherence to medical treatment recommendations for congenital adrenal hyperplasia.

Regulation of cell proliferation by protein tyrosine phosphatases (PTPs) suggests that PTPs are important tumor suppressor genes. The gene encoding the leukocyte common-antigen-related (LAR) PTP receptor maps to chromosome 1p32-33, a region in which loss of heterozygosity is associated with human pheochromocytoma and other neuroectodermal tumors. The rat pheochromocytoma PC12 cell line was originally derived from the transplantable P259 tumor originating from the New England Deaconess Hospital (NEDH) line of Wistar inbred rats. Compared with their Wistar counterparts, 1-2-year-old NEDH rats exhibit a high incidence of spontaneous pheochromocytomas. This study investigates whether levels of LAR transcripts and protein are altered in NEDH adrenal tissue prior to tumor onset. In addition, alternative splicing of an LAR extracellular domain [LAR alternatively spliced element-c (LASE-c)], regulating LAR interaction with extracellular matrix components, was examined. These changes in LAR expression and alternative splicing were hypothesized to be more pronounced in tumor tissue and PC12 cells. Northern blot analysis demonstrated the presence of the approximately 5 kb LAR transcript in all cell lines examined, except PC12. In adrenal medulla tissue harvested from 2-3-month-old rats, LAR approximately 8 and approximately 5 kb transcript expression was decreased in NEDH compared with Wistar samples. RT-PCR demonstrated increased splicing of the LASE-c 27 bp alternatively spliced insert in the LAR extracellular domain in NEDH adrenal medulla tissue. Even greater LASE-c splicing was detected in adrenal medulla tumor tissue derived from 12-month-old NEDH rats and in PC12 cells. Western blot analysis demonstrated decreased levels of LAR protein and increased levels of LASE-c containing LAR protein isoforms in NEDH adrenal medulla tissue. These studies demonstrate that patterns of altered LAR expression present in PC12 cells and in pheochromocytoma tumor tissue are also present in adrenal tissue predisposed to a high incidence of spontaneous pheochromocytoma.

Mn(2+) treatment has been shown to promote neurite outgrowth in rat pheochromocytoma (PC12) cells in a time- and dose-dependent manner. This process is mediated through the interactions of extracellular matrix (ECM) proteins and integrin receptors. Studies were performed to determine whether the phosphorylation of the MAP kinases, ERK1 and 2, is required for Mn(2+)-induced neurite outgrowth. A time- and dose-dependent increase in phosphorylation of both ERK1 and 2 was observed upon treatment of PC12 cells with Mn(2+). Phosphorylation of the ERKs occurred as early as 2 hr after initiating treatment, with a maximum increase occurring at approximately 24 hr. Inhibition of MEK with the specific inhibitor, PD98059, blocked the phosphorylation of ERK1 and 2 and increased Mn(2+) toxicity. When cells were grown in serum-free defined medium, Mn(2+)-induced phosphorylation of ERK1 and ERK2 occurred in cells grown on surfaces treated with growth serum or fibronectin but not on surfaces treated with poly-L-lysine. In addition, the pentapeptide GRGDS, which blocks RGD-mediated interactions, inhibited Mn(2+)-induced phosphorylation of ERK1 and 2. The Mn(2+)-induced increase in phosphorylated ERK1 and 2 was not seen in a PC12 cell line that does not respond to Mn(2+). These data support the hypothesis that integrin-mediated activation of the MAPK signal transduction pathway leading to the activation of ERK1 and 2 is required for Mn(2+)-induced PC12 differentiation and neurite outgrowth., (Copyright 1999 Wiley-Liss, Inc.)

Objective: An increased response of 17-hydroxyprogesterone to ACTH stimulation has been observed in adrenal incidentaloma and linked to an impairment of either 21-hydroxylase or of 11beta-hydroxylase activity. To analyse this question further, we investigated the steroidogenic pathways in a series of 17 adrenal incidentalomas., Design and Patients: 17 patients (7 women, 10 men; mean age, 62 +/- 12 years) with non-histologically analyzed adrenal incidentalomas were prospectively evaluated., Methods: The following variables were investigated: 24-h urinary methanephrines and free cortisol excretion; plasma levels of ACTH and dehydroepiandrosterone; overnight dexamethasone suppression test; 1-24 ACTH stimulation test with measurement of: cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, aldosterone, 11-deoxycorticosterone, progesterone, 17-hydroxypregnenolone, Delta4-androstenedione, dehydroepiandrosterone and 21-deoxycortisol., Results: Discordant features of subclinical hypercorticism were noted in one case. No patient had dehydroepiandrosterone sulfate levels in the normal range for his or her age. Peak 17-hydroxyprogesterone and peak 21-deoxycortisol disclosed impairment of 21-hydroxylase in 11 and 10 cases respectively. An increased 11-deoxycortisol/cortisol ratio identified reduced activity of 11beta-hydroxylase in 11 patients. Eight patients displayed features of mild 17,20-lyase impairment, which was related to 21-hydroxylase dysfunction. Whereas only 2 patients showed no enzyme modification, 9 displayed alterations of at least two pathways., Conclusion: In our hands, a combination of enzyme dysfunction was frequently observed. Shared biochemical mechanisms could explain combined 17,20-lyase and 21-hydroxylase alterations, whereas coexistence of 21-hydroxylase (particularly when based on peak 21-deoxycortisol) and 11beta-hydroxylase is more puzzling.

Bovine cholesterol side-chain cleavage cytochrome P450 (P450scc; product of the CYP11A gene) gene expression is regulated by gonadotropins via cAMP in the ovary, and by ACTH via cAMP in adrenal cortical cells. Previously, we characterized response elements located at -57/-32 and at -111/-101 bp in the 5'-flanking region of the bovine CYP11A gene required for cAMP-stimulated transcription in both mouse Y-1 adrenal tumor cells and bovine ovarian cells in primary culture, which bind SF-1 (or Ad4-BP) and Sp1, respectively. The role of these transcription factors in CYP11A transcription was further confirmed by deletion and mutation analyses. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites and a mammalian two-hybrid system indicate that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y-1 cells. Here we report that SF-1 and Sp1 are able to associate with one another in vitro and in vivo. The NH2-terminal region of SF-1, especially the DNA-binding domain, is the binding site for Sp1. In addition, as CBP is a common coactivator required for the transcriptional activity of numerous transcription factors including nuclear receptors, we investigated whether CBP functions as a cofactor for the regulation of bovine CYP11A promoter activity. We show here that CBP enhanced the PKA-induced CYP11A promoter activity, while a double mutation of both Sp1 and SF-1 sites within the CYP11A promoter region abolished CBP-induced activity. Furthermore, CBP stimulated Sp1-dependent transactivation, and a CBP/Sp1 complex in vivo was demonstrated by a co-immunoprecipitation assay. Also, CBP potentiated the transcriptional activity of GAL4-SF-1 in the presence of PKA. Thus, the cooperation between SF-1 and Sp1, required for the regulation of bovine CYP11A gene expression, is mediated by a direct protein-protein interaction and/or the common coactivator CBP.

Adrenarche is the maturational increase of adrenal androgens that takes place in 6-8 year old children. In order to study the role of 3 beta HSD in the regulation of the synthesis of human adrenal androgens, the abundance of 3 beta HSD mRNA (Dot Blot and semiquantitative RT-PCR) was measured in 11 human prepubertal and early pubertal adrenal tissues. Subjects were divided in 2 age groups (Gr): Gr1, < 8 years (y) old (n = 6, range 0.1-2.5) and Gr2, > or = 8 y old (n = 5, range 8.0-13.0). Tissue from one adrenal tumor with Cushing's syndrome (TSC) and 2 virilizing adrenal tumors (TV), as well as adrenal cells prepared from the TSC and from 1 TV were also studied. They were maintained in culture for 3 days in basal conditions (BC) and under ACTH and IGF-1 stimulation. mRNA in Gr1 was higher than in Gr2 (Dot blot: 4.65 +/- 2.70 and 0.28 +/- 0.27 AU, p = 0.006; RT-PCR: 21.5 +/- 12.5 and 6.77 +/- 3.78 AU, p = 0.039, respectively). 3 beta HSD mRNA in TSC (8.74 +/- 1.74) was higher than in the 2 TVs (0.47 +/- 0.02 and 0.87 +/- 0.08) p = 0.001. In TSC cells, basal mRNA (0.82 +/- 0.10) decreased under ACTH (0.55 +/- 0.06), p = 0.005, and increased under IGF-1 (2.36 +/- 0.07), p = 0.006. No changes were observed in TV cells. On day 3, TV cells in BC secreted 1170.0 +/- 210.0 and 335.0 +/- 29.0 pmol/10(6) cells in 24 hs of DHEAS and androstenedione, while TSC cells secreted 17.1 +/- 3.5 and 73.7 +/- 11.7, respectively. Values increased under ACTH in TV cells (2006.0 +/- 360.0 and 525.0 +/- 76.0) and in TSC cells (29.8 +/- 5.4 and 366.8 +/- 129) p < 0.05, but they decreased under IGF-1 only in TSC cells (7.9 +/- 2.4 and 43.7 +/- 6.1) p < 0.05. These data support the hypothesis that human adrenarche could be secondary to a decrease of 3 beta HSD mRNA. Our finding that when 3 beta HSD mRNA decreases androgen secretion increases (ACTH) and when 3 beta HSD mRNA increases androgen secretion decreases (IGF-1), strongly suggests that 3 beta HSD has a modulatory role in adrenal androgen steroidogenesis.

Histological analysis of surgically removed adrenal masses often fails to differentiate between benign and malignant tumors. In normal cells, the telomeric ends of the chromosomes are shortened with each cell division, leading to chromosome destabilization and cellular senescence after a critical number of cell cycles. In tumor cells, telomere shortening is prevented by a specific DNA polymerase, called telomerase. In an effort to clarify the role of telomerase in the pathogenesis of adrenal tumors, and to test whether its activity could serve as marker of malignancy, we measured telomerase activity in 41 human adrenal tissue samples that were classified both by the clinical course and by histological examination. Telomerase activity was determined by TRAP ELISA and expressed as high (>50% of positive control telomerase activity), medium (31-50%), low (11-30%), very low (< or = 10%), or absent (0%). The 8 normal adrenal tissue samples showed very low levels of telomerase activity. Mean telomerase activity also very low in 3/3 incidentalomas, 6/6 Cushing adenomas, 6/6 Conn adenomas, 7/7 adrenocortical carcinomas, 8/8 benign pheochromocytomas, and 2/3 malignant pheochromocytomas. In contrast, one malignant pheochromocytoma showed high telomerase activity. These data indicate that telomerase activity may not be a suitable marker for malignancy in the adrenal gland. Our results also challenge the current dogma of close correlation between cell dedifferentiation and telomerase activity.

Background: Telomerase is an enzyme that adds repeated telomere sequences to the ends of chromosome arms. It helps maintain both the length of telomere and infinite cell proliferation. In recent years, telomerase activity has been considered an important characteristic that differentiates between normal and cancerous cells. Because the authors often encountered difficulties in distinguishing between benign and malignant adrenal tumors, they investigated whether the expression of telomerase activity could distinguish potentially malignant adrenal tumors., Methods: The authors examined telomerase activity in 48 samples of adrenal tumor tissue and 27 samples of adjacent normal adrenal tissue. All samples were obtained from 48 patients who underwent surgery at Hamamatsu University Hospital in Hamamatsu, Japan. Based on the clinical and postoperative pathologic examinations, 45 samples were diagnosed as benign and 3 were diagnosed as malignant. Telomerase activity was examined using a telomerase repeat amplification protocol (TRAP) assay., Results: Of the 48 adrenal tumor samples, 7 (14.6%) had telomerase activity. All adjacent normal adrenal tissues were negative for telomerase activity. Of the telomerase positive samples, two were clinically known adrenocortical carcinoma, and another was metastatic adrenal tumor from lung carcinoma. Four other telomerase positive samples were diagnosed as benign after clinical and initial pathologic examinations. However, two of the patients from whom these samples were taken developed metastatic lesions after adrenalectomy., Conclusions: A telomerase assay of adrenal tumors may help predict their malignant potential.

Telomeres consist of short repeated sequences that are shortened on continuous cell proliferations and synthesized by telomerase, an RNA-dependent DNA polymerase. Recent molecular studies have reported that telomerase is activated in most human cancers, whereas it is not detected in most somatic cells. These findings indicate that the positive telomerase activity is closely related to the malignant potential of human tumors. In several types of human tumors, including adrenal cortical tumors and pheochromocytomas, it is very difficult to predict the malignant potential using conventional histopathologic examination. To determine whether telomerase activity is useful as a diagnostic marker, we examined telomerase activity in adrenal cortical tumors and pheochromocytomas with special reference to their clinicopathologic features. Using a highly sensitive polymerase chain reaction (PCR)-based detection method, telomerase activity was demonstrated in one of 13 adrenal cortical tumors and two of seven pheochromocytomas, whereas all seven normal portions of adrenal gland failed to showed any telomerase activity. Although none of the tumors examined in this study was associated with metastasis, these three telomerase-positive tumors were accompanied by clinicopathologic features suggesting malignant potential. Telomerase activity might be a potential marker for estimating the biologic characteristics of adrenal cortical tumors and pheochromocytomas.

Background: Although malignant pheochromocytoma is a life-threatening illness, there is no excretory profile that is predictive of malignancy. Reliable prediction of malignant behavior on the basis of histopathology is also notoriously difficult. Although DNA ploidy can be a helpful indicator, aneuploidy per se is not considered to be a specific marker of malignancy. Mutations in p53 have been reported to occur frequently in cases of benign pheochromocytoma. Thus, molecular analysis of this protein is not a useful diagnostic tool. The authors analyzed telomerase activity in benign and malignant pheochromocytomas and assessed its utility as a prognostic marker., Methods: Telomerase activity was estimated in 16 benign pheochromocytomas, 3 malignant pheochromocytomas, and 16 normal adrenal medullae by the fluorescence-based telomeric repeat amplification protocol. The telomerase activities of each group of samples were compared., Results: No telomerase activity was detected in any of the normal adrenal medullae or the benign pheochromocytomas. By contrast, all of the three malignant pheochromocytomas had elevated telomerase activity. The telomerase activity in each tumor was determined to be 87.6 units, 71.2 units, and 180.3 units, respectively., Conclusions: The expression of telomerase activity in pheochromocytoma clearly suggests the malignant behavior of the component cells. Analysis of telomerase activity appears to be a powerful tool for the diagnosis of malignant pheochromocytoma.

Hip-Arg-Phe-, Hip-Phe-Arg- and Hip-His-Leu-cleaving dipeptidyl carboxypeptidase activities were measured in the supernatant (S2) and pellet (P2) fractions obtained by ultracentrifugation of human adrenal tumor preparations. Negligible enzyme activity was found in cortical tumor whereas highly significant activities were present in the P2 fractions of the two pheochromocytoma specimens. The hydrolysis rates, expressed in terms of the percent of added substrate were 58-66%/60 min for Hip-Phe-Arg, 55-58%/60 min for Hip-Arg-Phe and 19-30%/60 min for Hip-His-Leu. The angiotensin-converting enzyme inhibitor, captopril, differentially inhibited the enzyme splitting Hip-His-Leu versus the one cleaving Hip-Arg-Phe; Hip-Phe-Arg is probably the substrate of both. It is concluded that the Hip-Arg-Phe-cleaving enzyme in adrenomedullary tumor is probably identical to the purportedly novel dipeptidyl carboxypeptidase that we detected earlier in rabbit ear artery wall, which converts (Met5)-enkephalin-Arg6,Phe7 to (Met5)-enkephalin.

To investigate the effect of neuronal differentiation on the capacity of antisense oligonucleotides (AS-ODNs) to suppress the production of acetylcholinesterase (AChE) in rat pheochromocytoma cells, we tested seven 3'-phosphorothioated AS-ODNs targeted to ACHEmRNA and two control ODNs. Three different administration protocols were used: oligonucleotides were added at 1 microM for 24 hours to nondifferentiated PC12 cells, together with nerve growth factor (NGF) or 24 hours following NGF-induced cholinergic differentiation. The content of free thiol groups in lysed cells was measured to evaluate cell number, therefore, survival, and the rate of acetylthiocholine hydrolysis was the measure of AChE activity. Among nondifferentiated cells, over 95% survived treatment with 8 of 9 of the ODNs. Moreover, two AS-ODN suppressed AChE activity in non-differentiated PC12 cells by 16%-20% as compared with 10% suppression by control ODNs (P < or = 0.01). When added concurrently with NGF, one other AS-ODN suppressed AChE activity significantly better (28%) than the control ODNs (16%). Moreover, when added following NGF treatment, which induced a significant increase in AChE activity, four different AS-ODNs but not the control ODNs suppressed 20%-35% of the enhanced AChE activity (p < or = 0.01). Reduced levels of AChE mRNA but no difference in actin mRNA levels were observed by following the kinetics of RT-PCR amplification in differentiated PC12 cells treated with these four AS-ODNs, as compared with control cells. Our findings demonstrate a differentiation-related increase in the susceptibility of PC12 cells to inhibition by specific AS-ODNs, suggesting the use of this model system to select AS-ODNs for suppression of AChE levels in the treatment of neurodegenerative diseases associated with cholinergic malfunction.

Recent reports have shown an exaggerated response of 17-hydroxyprogesterone in up to 70% of patients with incidentally detected adrenal adenomas ('incidentalomas'). This has been explained by pre-existing 21-hydroxylase deficiency which may be a pathogenetic factor in the development of adrenal tumours. However, other defects in steroidogenesis, such as mild 11 beta-hydroxylase deficiency, could also result in increased 17-hydroxyprogesterone secretion. We therefore studied the glucocorticoid and mineralocorticoid pathways in patients with adrenal 'incidentalomas' by measuring multiple adrenal steroids before and after 1-24 ACTH stimulation. Twenty patients with adrenal 'incidentalomas' (14 females, 6 males) and 27 healthy controls (14 females, 13 males) were studied. All subjects underwent a 1-24 ACTH stimulation test (250 micrograms i.v.) with determination of progesterone, 11-deoxycorticosterone, corticosterone, 17-hydroxyprogesterone, 11-deoxycortisol and cortisol at 0 and 60 min. All steroids were measured by RIA after extraction and HPLC. Patients with 'incidentalomas' had higher stimulated concentrations of 17-hydroxyprogesterone (21.6 +/- 8.4 vs 4.2 +/- 0.3 nmol/I; P < or = 0.001), 11-deoxycortisol (8.1 +/- 1.2 vs 3.6 +/- 0.3 nmol/I; P < or = 0.001), progesterone (8.28 +/- 2.82, vs 1.08 +/- 0.15 nmol/I; P < or = 0.001), and 11-deoxycorticosterone (2.1 +/- 0.39 vs 0.78 +/- 0.12 nmol/I; P = 0.002) compared with controls. In contrast, cortisol and corticosterone concentrations were not different. There was evidence for impairment of 11 beta-hydroxylase activity by an increased 11-deoxycortisol/ cortisol ratio (0.012 +/- 0.003 vs 0.005 +/- 0.001 in controls; P = 0.002) and 11-deoxycorticosterone/ corticosterone ratio (0.04 +/- 0.003 vs 0.015 +/- 0.003; P = 0.003). The conclusions reached were that patients with adrenal 'incidentalomas' have increased responses of precursors of the mineralocorticoid and glucocorticoid pathway including 17-hydroxyprogesterone after stimulation with ACTH. This seems to be caused by impairment of 11 beta-hydroxylase activity rather than by impaired 21-hydroxylase activity in these tumours.

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the interconversion of cortisol (F) to inactive cortisone (E) in man (corticosterone (B) to 11-dehydrocorticosterone (A) in rodents) and plays a crucial role in regulating corticosteroid hormone action. Two isoforms of this enzyme have been characterized; a low affinity NADP(H)-dependent enzyme (11 beta-HSD1) and a high affinity NAD-dependent dehydrogenase (11 beta-HSD2). We have analysed the expression of 11 beta-HSD in the rodent and human adrenal gland and have investigated its role with respect to glucocorticoid-mediated catecholamine biosynthesis. Our studies indicated higher expression of 11 beta-HSD2 mRNA in male versus female intact mouse adrenal. Both 11 beta-HSD isoforms were detected in intact male rat adrenal homogenates. For the 11 beta-HSD1 isoform, NADPH-dependent oxo-reductase activity exceeded that of NADP-dependent dehydrogenase activity (188 versus 98 pmol/mg.protein.hr). In situ hybridisation studies indicated specific localisation of 11 beta-HSD1 mRNA to cells at the corticomedullary junction. 11 beta-HSD2 mRNA was uniformly distributed across the cortex and was low/absent in the medulla. Administration of glycyrrhizic acid in vivo (> 100 mg/kg for 4 days) resulted in inhibition of 11 beta-HSD1 mRNA and activity and a decrease in mRNA levels for the glucocorticoid-dependent enzyme, phenylethanolamine N-methyltransferase, whilst levels of the glucocorticoid-independent enzyme, tyrosine hydroxylase were unchanged. No 11 beta-HSD expression was observed in the rat phaeochromocytoma cell line, PC12 cells, nor in human normal adrenal gland or phaeochromocytoma specimens. There are marked species and sex differences in the expression of 11 beta-HSD isoforms within the adrenal. The role of 11 beta-HSD within the adrenal gland remains obscure, but at least in the rat, the expression of the reductase enzyme, 11 beta-HSD1, to the corticomedullary junction may serve to maintain high medullary glucocorticoid concentrations required for catecholamine biosynthesis.

Tyrosine hydroxylase (TH) gene transcription rate is increased in rat adrenal medulla after administration of muscarinic agonists. In order to study this muscarinic regulation of TH gene expression in more detail, we have generated a rat pheochromocytoma PC18 cell line that stably expresses the mouse m1 muscarinic acetylcholine receptor. Treatment of this cell line, designated PC18/m1-13, with carbachol leads to rapid increases in phosphatidylinositol turnover and intracellular [Ca2+]i; these increases are totally blocked by the muscarinic antagonist atropine. Carbachol produces no changes in cAMP levels or protein kinase A activity in PC18/m1-13 cells. TH mRNA levels in PC18/m1-13 cells increase approximately 3-fold after 6 h of treatment with carbachol. This induction of TH mRNA is also completely inhibited by simultaneous treatment with atropine. Transient transfection assays using a TH gene promoter-chloramphenicol acetyltransferase (TH-CAT) construct demonstrate that sequences within the most proximal 272 bp of the TH gene 5'-flanking region are responsive to carbachol in PC18/m1-13 cells. Studies using TH-CAT constructs with site-directed mutations within the TH gene promoter indicate that the responsiveness of the promoter to carbachol is mediated primarily by the cAMP response element; however, the AP1 site also participates to a lesser extent in this response. The carbachol-mediated stimulation of TH gene promoter activity is partially inhibited by down-regulation of protein kinase C (PKC) or by treatment with the Ca2+/calmodulin-dependent protein kinase inhibitor, KN62. These results are consistent with the hypothesis that agonist occupation of m1 muscarinic receptors stimulates the TH gene via signal transduction pathways that are initiated by activation of PKC and Ca2+/calmodulin-dependent protein kinase, leading to activation of transcription factors that interact with the TH CRE and AP1 sites.

Bilateral adrenal aldosterone-producing adenomas (APA) are rare. It is important to distinguish bilateral APA from idiopathic hyperaldosteronism (IHA) which is due to bilateral hyperplasia of the adrenal cortex. We present two patients with bilateral APA in whom the diagnosis was made histochemically by analyzing steroidogenic enzymes. They showed hypokalemia, high plasma aldosterone concentration (PAC) and suppressed plasma renin activity (PRA). Bilateral adrenal tumors were represented by computed tomography, and surgical resection was performed. In both cases, cytochrome P-450 and other enzymes that were involved in aldosterone synthesis were found mainly in tumor, but little in the zona glomerulosa of the adjacent adrenals, which showed paradoxical hyperplasia. Such cases are difficult to distinguish from IHA. The two disorders were differentiated by immunohistochemical analysis of steroidogenic enzymes.

We performed a comparative study of catecholamine content, tyrosine hydroxylase (TH) activity, and TH mRNA levels in normal human adrenals and various clinical forms of human pheochromocytoma. We studied sporadic, benign intra-adrenal chromaffin tumors and other non-malignant intra-adrenal tumors associated with multiple endocrine neoplasia type 2B (MEN 2B) and von Hippel-Lindau disease along with one extra-adrenal malignant pheochromocytoma. Our findings suggest substantial differences in TH transcriptional rates or the stability of TH mRNA or both may contribute to altered TH expression in human chromaffin cells associated with "normal" adrenal tissues and various forms of pheochromocytoma and distinctive patterns of expression in the different settings in which these tumors arise.

Aromatic L-amino acid decarboxylase (AADC) is found in both neuronal cells and nonneuronal cells, and a single gene encodes rat AADC in both neuronal and nonneuronal tissues. However, two cDNAs for this enzyme have been identified: one from the liver and the other from pheochromocytoma. Exons 1a and 1b are found in the liver cDNA and the pheochromocytoma cDNA, respectively. In the third exon (exon 2), there are two alternatively utilized splicing acceptors specific to these exons, 1a and 1b. Structural analysis of the rat AADC gene showed that both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. To demonstrate whether alternative promoter usage and splicing are tissue specific and whether the exons 1a and 1b are differentially and specifically transcribed in nonneuronal and neuronal cells, respectively, in situ hybridization histochemistry for the rat brain, adrenal gland, liver, and kidney was carried out using these two exon probes. The exon 1a probe specifically identified AADC mRNA only in nonneuronal cells, including the liver and kidney, and the exon 1b probe localized AADC mRNA to monoaminergic neurons in the CNS and the adrenal medulla. Thus, both alternative promoter usage and differential splicing are in fact operative for the tissue-specific expression of the rat AADC gene.

Exposure of PC12-VG cells to an extremely low frequency magnetic field (ELFMF) enhanced the beta-galactosidase gene expression stimulated by treatment of the cells with forskolin. The enhancing effect of the ELFMF was inhibited by treatment of the cells with a specific inhibitor of PKC, calphostin C, as well as with the Ca2+ entry blockers nifedipin and dantrolen. Enhancement appeared within the first hour of a 4h forskolin treatment when the ELFMF was given at different times during culture. We speculate that exposure of PC12-VG cells to an ELFMF during the early response to forskolin treatment affects cell signal transduction, resulting in enhanced gene expression.

Based on the cDNA sequence of the human and bovine endothelin converting enzyme (ECE-1) we have developed a novel reverse transcription polymerase chain reaction to investigate the tissue expression of this gene. We were able to specifically detect the gene mRNA starting from very limited amount of tissue in all human as well as bovine tissues examined. Thus, our results confirm a widespread expression of the ECE-1 gene in human tissues, in keeping with the findings in other species, and suggest a major biological role of ECE-1.

Pheochromocytomas synthesize and release catecholamines, which subsequently are related to various clinical manifestations of the disease. However, pheochromocytomas are not innervated and the catecholamine release and synthesis are not initiated by neural impulses. It is still unknown how catecholamine synthesis is regulated in pheochromocytomas. As a first step toward understanding the molecular mechanisms by which catecholamine synthesis is controlled in the tumor, we measured the levels of mRNA coding for the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) and catecholamines in 6 pheochromocytomas and 2 normal adrenal glands. The TH mRNA level was overexpressed and the catecholamine contents were high in 4 out of 6 pheochromocytomas. There was a close correlation between the TH mRNA level and the catecholamines content in the tumors. We also examined the gene expression of the messengers of other catecholamine synthesizing enzymes, dopamine beta-hydroxylase (DBH) and aromatic 1-amino acid decarboxylase (AADC) in pheochromocytomas. The expression of these genes was in parallel with that of TH mRNA in the tumors. These findings indicate that catecholamine overproduction in pheochromocytomas is mediated by the overexpression of genes coding for catecholamines synthesizing enzymes, TH, DBH, and AADC.

There are almost no special histopathological characteristics or criteria that exactly define a malignant pheochromocytoma. Tissue concentrations of catecholamine metabolites and superoxide dismutase activity have been proposed as possible candidates for discriminating between benign and malignant pheochromocytomas. Tissue concentrations of dihydroxyphenylalanine, metanephrine, normetanephrine, vanillylmandelic acid, and 3-methoxy-4-hydroxyphenylethylglycol were determined in 29 normal adrenal medullas, 13 benign pheochromocytomas and 6 malignant pheochromocytomas, respectively. The copper-zinc superoxide dismutase and manganese superoxide dismutase activities in remnants of these tissues were determined by interruption of nitric formation from hydroxylamines. Catecholamine metabolites and copper-zinc superoxide dismutase activity in benign and malignant pheochromocytomas were identical. Manganese superoxide dismutase activity in malignant pheochromocytoma was the lowest among the groups examined. These data suggest that the assay of catecholamine metabolites in removed specimens is not a reliable method for making a differential diagnosis of benign or malignant pheochromocytoma. However, a low level of manganese superoxide dismutase activity in malignant pheochromocytoma may be a marker for malignancy of this neoplasm.

Human tyrosine hydroxylase isoform 1 (hTH1) was expressed in Escherichia coli, purified as the apoenzyme, and reconstituted with iron. The resonance Raman spectra of hTH1 complexed with dopamine, noradrenaline, tyramine, and catechol have been studied and compared to those obtained for TH isolated from bovine adrenal glands or rat phaeochromocytoma tissue. A TH-phenolate complex is reported for the first time. Using dopamine selectively 18O-labeled in the 3-position or both 3- and 4-hydroxy positions, we have been able to assign unambiguously the origin of the low-frequency vibration bands: the band at 631 cm-1 involves the oxygen in the 4-position; the band at 592 cm-1 involves the oxygen in the 3-position, and the band around 528 cm-1 is shifted by both, suggesting a chelated mode vibration. A small shift of the 1275 cm-1 band and no shift of the 1320 cm-1 band were observed, showing that those two bands involve essentially ring vibrations of the catecholate moiety, rather than the C--O stretching vibration as previously suggested. The spectrum of the catechol-d6-hTH1 complex confirms this assignment. The resonance Raman spectra of the 54Fe, 56Fe, or 57Fe isotope-containing enzymes complexed with dopamine are virtually identical, showing that the component of the iron in the approximately 600 cm-1 vibrations is too small to be observed. These results provide a better understanding of the Raman properties of iron-catecholate complexes in this enzyme, as well as in other metalloproteins and model compounds.

Telomerase activity was analysed in 100 neuroblastoma cases. Although telomerase activity was not detected in normal adrenal tissues or benign ganglioneuromas, almost all neuroblastomas (94%) did express it, suggesting an important role for telomerase in neuroblastoma development. Neuroblastomas with high telomerase activity had other genetic changes (for example, N-myc amplification) and an unfavourable prognosis, whereas tumours with low telomerase activity were devoid of such genetic alterations and were associated with a favourable prognosis. Three neuroblastomas lacking telomerase activity regressed (stage IVS). Thus telomerase expression may be required as a critical step in the multigenetic process of tumorigenesis, and two different pathways may exist for the development of neuroblastoma.

A novel protein [designated protein tyrosine phosphatase-like protein (PTPLP)] which is distantly related to receptor-type protein tyrosine phosphatases (PTPases) was cloned from a rat pheochromocytoma cell line. The PTPLP was detected exclusively in the brain. Overexpression of the PTPLP decreased the basal PTPase activity of COS-7 cells for Raytide. These results suggest that PTPLP may function as a negative regulator of PTPases in neuronal tissues.

To elucidate the mechanisms of abnormal steroid production in hyperfunctioning and non-hyperfunctioning adrenal tumors, we examined both the activities and amounts of steroidogenic cytochromes P450 in the tumor and non-tumor portions of these adrenals at the posttranslational (protein) level. Adrenals from 5 patients with primary aldosteronism, 5 with Cushing's syndrome, 1 with deoxycorticosterone (DOC)-producing adenoma, 10 with non-hyperfunctioning adrenal adenoma, and 5 subjects with normal control adrenals (obtained from patients with renal cell carcinoma) were used in our studies. Activities of P450scc, P45011 beta and P450aldo, and P450C21 and P-45017 alpha were assayed in a reconstituted enzyme system using 20 alpha-hydroxycholesterol, DOC, and progesterone, respectively, and the substrate and the extracted products were analyzed by HPLC. Enzyme amounts were determined by immunoblot analysis with anti-bovine P450scc, P45011 beta, and P450C21 IgG, and anti-porcine P45017 alpha IgG. Human P450aldo was only detected in the tumor portion of primary aldosteronism adrenals, with both activities and amounts of other P-450s similar to those in the non-tumor portion of primary aldosteronism and normal controls. In Cushing's syndrome, both activities and amounts of P45017 alpha and P450C21 were significantly increased in the tumor compared with those in the non-tumor portion of Cushing's syndrome and normal controls. In DOC-producing adenoma, both activities and amounts of P45017 alpha and P45011 beta in the tumor portion of the adenoma decreased compared with normal control, while those of other P450s were similar to normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Among catecholamine synthesizing enzymes, phenylethanolamine-N-methyltransferase (PNMT) exists only in adrenal and extra-adrenal pheochromocytoma has been believed not to produce adrenaline. However, adrenaline production is clinically active in some extra-adrenal cases. To investigate this controversy, the localization of catecholamine synthesizing enzymes was studied by immunohistochemical staining in extra-adrenal and adrenal cases. The results showed that PNMT was expressed not only in many cases of adrenal pheochromocytoma, but also in extra-adrenal pheochromocytoma. It is suspected that little adrenaline production in extra-adrenal cases is attributable to a lack of a good environment for the activation of PNMT by a high concentration of glucocorticoids.

In a girl aged 5 years with a virilizing adrenal adenoma the urinary and plasma steroid findings suggested the diagnosis of congenital adrenal hyperplasia due to P450c11 (11 beta-hydroxylase) deficiency. After removal of the tumour clinical signs receded and the hormonal values normalized. RNA analysis of the tumour tissue revealed low amounts of P450c11 mRNA which indicates that P450c11 deficiency of the adenoma caused the steroid abnormalities in this girl.

The intracellular localization of tyrosine hydroxylase (TH), which is the rate limiting enzyme in catecholamine (CA) biosynthesis, and its activity in various adrenal and other neuroendocrine tumors was studied. TH was strongly localized in adrenal medulla, pheochromocytoma, and paraganglioma, but was scatteredly expressed in neuroblastoma. TH was not detected in adrenocortical tumors, ganglioneuroma, and other neuroendocrine tumors. Neuron specific enolase (NSE) was found in all neuroendocrine tumors, but Grimelius staining showed only the secreting granules of the tumor cells. TH activity was significantly high in pheochromocytoma and paraganglioma as compared with that in normal adrenal gland, whereas TH activity was low in a neuroblastoma and was undetectable in other tumors. These findings indicate that TH correlates well with the biosynthetic function of CA in the tumor cell and, thus, both the immunostaining of TH and the measurement of its activity in adreno-medullary and related tumors may provide some information about the process of cell differentiation in these tumors.

Expressions of two Kex2-related proteases, Pc2 and PC1/PC3, and of one of their possible substrates, proenkephalin, were examined in normal (n = 7) and various pathological (n = 48) human adrenal tissues. Northern blot analysis detected the expression of these genes in pheochromocytomas only. In the 20 pheochromocytomas studied with this technique, PC2, PC1/PC3 and proenkephalin were expressed in 85%, 50% and 90%, respectively. The presence of PC2 and PC1/PC3 was further confirmed using the sensitive RT/PCR techniques. Other evidence of human tumoral adrenal medullary PC2 expression was provided by in situ hybridization and immunohistochemistry. In addition, proenkephalin was expressed only in the pheochromocytomas expressing PC2 and/or PC1/PC3. These results demonstrate that functional Kex2-related endoproteases are expressed in human pheochromocytomas and may be involved in the processing of proenkephalin.

We studied the steroidogenetic action and concomitant subcellular redistribution of protein kinase C (PKC) in cortisol hypersecreting adrenal adenoma cells obtained from two patients with Cushing's syndrome. Isolated adenoma cells were treated with 10(-9) M and 10(-6) M 12-O-tetradecanoyl phorbol-13-acetate (TPA) or 10(-6) M ACTH. Treatment of the isolated adenoma cells with TPA resulted in cortisol secretion equivalent to that with ACTH-treatment. Immunoblot analysis of PKC during treatment with ACTH or TPA showed that PKC beta translocated from cytosol to membrane. A small amount of PKC alpha, but not membrane-associated PKC alpha, was detectable in the cytosolic fraction. It appeared that TPA-induced cortisol secretion mimicked ACTH-induced cortisol secretion, and that PKC beta translocated from cytosol to membrane on stimulation by both ACTH and TPA. We suggested that ACTH-induced cortisol secretion in human cortisol hypersecreting adrenal adenoma is mediated by PKC beta activation.

Stepwise selection in increasing H2O2 concentrations was used to obtain a PC12 cell variant designated HPR. This variant was stably resistant to H2O2 as compared with the parental PC12 cell line. HPR cells responded to nerve growth factor (NGF) by further enhancing H2O2 resistance. This variant was subcloned by limiting dilution to obtain the line referred to as HPR-C, which was stably resistant to H2O2 toxicity and retained NGF responses, including morphologic changes and further reduction of H2O2 toxicity. When compared with the parental PC12 line, the HPR-C subclone did not have higher levels of catalase or glutathione peroxidase (GSH Px) activity or mRNA expression (as assessed by PCR analysis of cDNA reverse transcribed from total cellular RNA). HPR-C cells retained the ability to respond to NGF treatment by increasing catalase and GSH Px activity and expression. These data suggest that the protective effects of conditioning lesions, unlike those of neurotrophins, are in part independent of changes in the activity or expression of antioxidant enzymes.

Aromatic L-amino acid decarboxylase (AADC) decarboxylates both L-5-hydroxytryptophan to serotonin in serotonergic neurons and pineal cells, and L-dopa to dopamine in catecholaminergic neurons and adrenal medullary cells. Thus AADC produces two major mammalian neurotransmitters and hormones. We isolated and sequenced a full-length, neuronal-type, cDNA encoding human AADC. It consisted of 1932 bases containing an open reading frame encoding 480 amino acids residues with a molecular weight of 53,891. We expressed a recombinant human AADC in COS cells and proved that the expressed enzyme decarboxylated both L-5-hydroxytryptophan to serotonin and L-dopa to dopamine. We have cloned genomic DNA of human AADC and determined the structure. The genomic DNA of human AADC consists of 15 exons spanning about 100 kilobases and exists as a single copy in the hapoloid genome. We have mapped the gene to chromosome band 7p12.1-p12.3 by fluorescence in situ hybridization. We cloned the nonneuronal type cDNA from human liver and identified another first exon different from the neuronal type cDNA. This showed that an alternative usage of the first exon produced two types of mRNAs in AADC and suggested that alternative splicing would regulate the tissue-specific expression of AADC.

Human tyrosine hydroxylase (HTH) RNA undergoes alternative splicing, and four different forms of HTH mRNA have been previously identified. Rabbit antibodies were raised against octapeptides unique to each of the four isoforms of HTH predicted from these mRNAs. Blot immunolabeling of human adrenal medulla, pheochromocytoma, and several neuroblastoma cell lines with affinity-purified anti-HTH peptide antibodies demonstrated the presence of all four HTH isoforms in each of these tissues. Quantitative immunolabeling assays for HTH-1, -2, and -4 were established, and HTH isoform levels were determined in several human neuroblastoma cell lines. Whereas total HTH levels differed up to fourfold among the HTH-positive neuroblastoma cell lines studied [LA-N-1, LA-N-5, CHP-234, BE(2)-C, and BE(2)-M17], the relative abundances of HTH isoforms in each of the cell lines were similar. Immunocytochemical analyses demonstrated that HTH immunoreactivity was distributed unequally among the cells in each of these neuroblastoma lines, and morphological interconversion did not account for this heterogeneity. A direct relationship between the percentage of HTH-positive cells and overall HTH levels was also observed. This relationship, in the absence of an apparent clonal basis for the heterogeneity, suggests that HTH expression in neuroblastoma cells may be controlled in a relatively "all-or-none" (bimodal) fashion.

Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days. The enzyme sedimented with an S(eo),w value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component. Two-dimensional gel isoelectric focusing/electrophoresis and tryptic digestion revealed that the heterogeneity could be accounted for by limited proteolysis of the 62 kDa component and the presence of covalently bound phosphate. The enzyme had a strong blue-green colour (epsilon 700 = 3.1 +/- 0.2 mM-iron-1.cm-1). The resonance Raman spectrum obtained with lambda excitation = 605 nm revealed the presence of an Fe(III)-catecholamine complex in the isolate enzyme, similar to that observed in the bovine adrenal enzyme [Andersson, Cox, Que, Flatmark & Haavik (1988) J. Biol. Chem. 263, 18621-18626]. In the rat PC12 enzyme, all of the iron present (0.53 +/- 0.03 atom per subunit) seems to be chelated by the feedback inhibitors (0.49 +/- 0.05 mol of dopamine and 0.10 +/- 0.03 mol of noradrenaline per mol of subunit). The e.p.r. spectra at 3.6 K show g-values at 7.0, 5.2 and 1.9 as observed for other catecholate-complexed enzymes. After phosphorylation of serine-40 and addition of L-tyrosine a new rhombic (magnitude of E/D = 0.33) e.p.r. species could be observed. Phosphorylation of serine-40 by cyclic AMP-dependent protein kinase increased the catalytic activity; depending on assay conditions, up to 80-110-fold activation could be observed when measured at high TH (i.e. high endogenous catecholamine) concentration.

Salsolinol is one of the dopamine-derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood-brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring cofactor of tyrosine hydroxylase, L-erythro-5,6,7,8-tetrahydrobiopterin, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using L-erythro-5,6,7,8-tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C-1 played an important role in changing the affinity to L-tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.

The levels of microsomal cytochrome P-450, steroidogenesis and microsomal and cytosolic epoxide hydrolase activities in normal human adrenal tissue (obtained from adult kidney transplant donors and autopsy material) and corresponding hyperplasia, adenomas and carcinomas (surgical biopsies) were determined. The increased steroid production demonstrated by most of the pathological tissue samples examined here was associated with either an unchanged or dramatically decreased specific microsomal content of cytochrome P-450. Furthermore, specific microsomal epoxide hydrolase activity was also found to be reduced in adrenocortical carcinomas, while the corresponding cytosolic activity was also decreased in at least two of these carcinomas. It is of interest to note in this connection that the level of microsomal epoxide hydrolase in slightly atropic adrenal cortex surrounding adrenocortical carcinomas was also found to be reduced. This would indicate that despite its appearance, this surrounding tissue is not normal in all respects. Thus, adrenocortical carcinomas fit into the common pattern in that their specific contents of microsomal cytochrome P-450 are dramatically decreased, but the simultaneous decrease in their microsomal epoxide hydrolase activity is more unusual.

The activity of alpha-galactosyltransferase in cultured rat pheochromocytoma subcloned (PC12h) cells was examined using Gb3 as the acceptor for the galactose from UDP-galactose. The major reaction product was identified as gal alpha 1-3Gb3 based on its mobility on thin-layer chromatographic (TLC) plates and susceptibility to specific galactosidases. The enzyme activity in PC12h cells was the highest at pH 7.0 while the presence of Triton CF-54 (0.1%) and Mn2+ (5 mM) was required for its full activity. The apparent Km values for Gb3 and UDP-galactose were 57 and 17 microM, respectively. The enzyme activity in PC12h cells was compared with that in parent PC12 cells, in which gal alpha 1-3Gb3 is not expressed in an appreciable amount. In the enzyme reaction with exogenous Gb3, the enzyme activity in PC12h cells was about 1.5-fold higher than that in PC12 cells. In the absence of exogenous Gb3, this difference became even more pronounced; gal alpha 1-3Gb3 was generated from endogenous Gb3 at a much higher rate in PC12h cells than in PC12 cells. These findings suggest that the higher level of the alpha-galactosyltransferase activity in PC12h cells may, at least in part, be responsible for the accumulation of unique neutral glycosphingolipids having gal alpha 1-3 terminal residues in the cells.

Patient 1 (a 50-year-old woman) with bilateral adrenocortical adenomas causing Cushing's syndrome underwent right-sided adrenalectomy. After an interval of 4 years 5 months, she underwent left-sided tumorectomy. Patient 2 (a 46-year-old woman) underwent bilateral tumorectomy after a frozen-section diagnosis. Each adrenal contained a single macronodule that consisted of compact cells, clear cells, and oxyphilic cells, which had characteristic enzyme histochemical and ultrastructural features. In addition, the adrenals had a so-called pigmented nodule and extracapsular small focus of compact and clear cell proliferation in case 2. The nonnodular part of the cortex was atrophic in both cases. Fourteen patients (mean +/- SD age, 42.5 +/- 5.55 years) with bilateral adrenocortical adenomas associated with Cushing's syndrome have been described in the literature. Thirteen (93%) of the patients were female. Three patients had unilateral or bilateral multiple adenomas. While the earlier described patients underwent bilateral total adrenalectomy, those in recent reports had part of the adrenal resected unilaterally or bilaterally (ie, tumorectomy). The above features are useful in differentiating bilateral adrenocortical adenoma from other types of bilateral adrenocortical lesions.

Immunohistochemical localization of the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), was investigated in 70 cases of functioning and non-functioning phaeochromocytomas comprising 52 of adrenal and 18 of extra-adrenal origin. Of 59 functioning tumours, 30 were mixed epinephrine and norepinephrine-producing (mixed type) and 29 were norepinephrine-producing tumours. TH, AADC and DBH were detected in all functioning phaeochromocytomas, but PNMT was limited to the mixed-type phaeochromocytomas. Non-functioning phaeochromocytomas were divided into two groups, comprising a complete type, which induced neither elevated plasma catecholamines nor their metabolites in urine, and an incomplete type which exhibited no elevated plasma catecholamines, but showed a slightly high urinary vanillylmandelic acid level. In the non-functioning complete-type tumours, immunoreactive TH was negative, but the incomplete tumours of the adrenal medulla had all four enzymes, and corresponded to a mixed-type phaeochromocytoma. AADC and DBH were present universally in all functioning and non-functioning tumours, including TH-negative tumours. TH is a rate-limiting enzyme of catecholamine biosynthesis and deficiency of TH is an important feature of extra-adrenal non-functioning phaeochromocytomas.